Molecular Evolution of Neuropeptide Y Receptors in Vertebrates

Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1019

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ERIK SALANECK

ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001 This thesis is based upon the following papers, which will be referred to by their Roman numerals:

I. SALANECK, E., HOLMBERG, S. K., BERGLUND, M. M., BOSWELL, T. AND LARHAMMAR, D. (2000) Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2. FEBS Letters 484:229-234.

II. SALANECK, E., FREDRIKSSON, R., LARSON, E.T., CONLON, J.M. AND LARHAMMAR, D. (2001) A neuropeptide Y receptor Y1-like gene from an agnathan, the European river lamprey: a potential ancestral gene (Submitted)

III. SALANECK, E., LARSON, E.T., AND LARHAMMAR, D. (2001). Three Neuropeptide Y receptors in the Y1 subfamily from the spiny dogfish, Squalus acanthias, support genome duplications in early vertebrate evolution (Submitted)

IV. LUNDELL, I., BERGLUND, M.M., STARBÄCK, P., SALANECK, E., GEHLERT, D.R. AND L ARHAMMAR, D. (1997) Cloning and Characterization of a Novel Neuropeptide Y Receptor Subtype in the Zebrafish. DNA Cell Biol, 16(11):1357-63.

Reprints were made with permission from the publishers: Paper I, Elsevier Science, Paper IV, Mary Ann Lieber Inc. TABLE OF CONTENTS

ABBREVIATIONS 3 INTRODUCTION 4 EVOLUTION AND THE VERTEBRATES 5 Birds 6 Advanced Bony Fishes 8 Sharks 9 Lampreys 10 MOLECULAR EVOLUTION 11 GENE DUPLICATIONS 12 NEUROSCIENCE AND G-PROTEIN COUPLED RECEPTORS 14 THE NPY PEPTIDE FAMILY 16 NPY 17 PYY and PMY 18 PP 18 NPY RECEPTORS 19 Y1 21 Y2 21 Y4 23 Y5 23 Y6 24 Ya and Yb/c 24 NPY receptors in invertebrates 25 RESEARCH AIMS 26 PAPER SUMMARIES: RESULTS AND CONCLUSION 27 Paper I 27 Paper II 27 Paper III 28 Paper IV 30 DISCUSSION 31 Chicken Y2 31 The teleost receptors 31 The Y1 subfamily in sharks 32 The lamprey Y receptor 33 NPY receptors in vertebrates 34 FUTURE PERSPECTIVES 36 ACKNOWLEDGMENTS 39 REFERENCES 40

2 ABBREVIATIONS aa amino acid Ca++ calcium ion cAMP cyclic adenosine-mono-phosphate ch chicken (Gallus gallus) cDNA complementary DNA CNS central nervous system DNA deoxyribonucleic acid gDNA genomic DNA GPCR G-protein coupled receptor HOX homeobox HSA Homo sapiens iPCR inverse PCR KO knock-out Lf Lampetra fluviatilis Leu leucine mRNA messenger RNA MP maximum parsimony MYA million years ago NA noradrenaline NJ neighbor joining NMR nuclear magnetic resonance NPY neuropeptide tyrosine p porcine PCR polymerase chain reaction PMY peptide methionine tyrosine Pro proline PY polypeptide tyrosine PYY peptide tyrosine tyrosine PP pancreatic polypeptide RNA ribonucleic acid RT reverse transcriptase SSC Sus scrofa Sq Squalus acanthias TM transmembrane z zebrafish

3 INTRODUCTION

n 1858 Charles Darwin, together with Alfred Russell Wallace, presented a joint paper at the Linnéan Society in London which proved to be one Iof the most important contributions to the field of science (Darwin & Wallace, 1858). The Origin of Species published one year later (Darwin, 1859) profoundly influenced the science of life, and the view of our own origins and lives. Although the theory of evolution must be accredited to Darwin, at the time of its presentation it lacked a mechanistic explanation. Not until the beginning of the 20th century was the work performed by Gregor Mendel assimilated into evolutionary biology. His investigation of the inheritance and genetics of the pea (Picea sativa) supplied the mechanism for the gradual change of species postulated by Darwin more than 50 years earlier. Ironically, the work on the basics of genetics by Mendel was performed almost simultaneously with the publishing of Darwin’s book, but did not at the time reach a wide scientific audience. A third breakthrough in the understanding of life and the mechanisms by which it evolves was made by Watson and Crick 1953, who described the structure of DNA (Watson & Crick, 1974); the molecule comprising the genes described by Mendel and the blueprints upon which Darwin’s natural selection exerts its effects. These blueprints comprised of four basic molecules denoted A, G, C and T could be read like a book not only by the reading systems of the cell, but also by biologists. Combinations of the four bases in the DNA are translated into sequences of amino acids, building proteins, the actual building blocks and machines used by cells and organisms. Evolution had previously been inferred primarily from studies of anatomy and development of organisms. After the discovery of the structure of DNA it became possible to measure the genetic differences between species quite simply by the differences in a gene. In this way the effects of natural selection could be measured in an objective manner, paving the way for the field of molecular evolution. Equipped with the means to investigate the relationships between all living organisms, it became possible for scientists to attempt reconstructing the paths that have lead to life as we know it. This thesis describes a contribution to the further understanding of evolution. Comparative studies were carried out for a group of neurophysiologically important molecules, the neuropeptide Y receptors, and the genes coding for these, in a number of vertebrate species. Thereby,

4 new insights were obtained regarding the evolutionary processes that formed these genes. By understanding the origins of these genes and the pathways they have followed during the past five hundred million years in diverse species, we may also contribute to the understanding of the physiological roles of neuropeptide Y in mammals, including ourselves. The sequence comparisons may potentially facilitate improved drug design for treatment of several pathological conditions.

EVOLUTION AND THE VERTEBRATES

Evolution means descent with modification or change of appearance, behavior, physiology or pattern of development over the course of generations. Evolution occurs by means of natural selection upon the inherited traits, or adaptations, owned by an individual organism. The individual with the best adaptations has the greatest likelihood of surviving and reproducing, hence passing its genes and adaptations on to the next generation. The evolutionary term fitness describes the average number of viable offspring from an individual, reflecting the evolutionary quality of an individual genome. Because sexual reproduction involves the mixing of genetic information from two individuals, individual offspring own distinct repertoires of genetic information from the parents. Natural selection acts upon the siblings, giving the best adapted sibling the highest likelihood of surviving and passing its, and thereby its parents’ genes on to the next generation. By these means the best combination of genes in a population will accumulate shaping the appearance and behavior of a species. Genetic information changes. Each and every time a cell divides all genetic material is copied so that each new cell obtains a full set of genes. Both during division of somatic cells, i.e. the cells comprising the body of an organism, as well as gamete cells, that will become sperm or egg cells, faults in the copying process or reshuffling of genetic information can occur. Any change or alteration in the genetic material is called a mutation. Since mutations occur at random, they are almost always of negative value to the gene product and only very seldomly neutral, i.e. not effecting the function of the gene product. Even though this process proceeds at random, enough neutral or at least nearly neutral changes to genetic information occur to generate the diversity that natural selection can act upon. It is thought that most mutations although neutral can become of value if the habitat or environment of the organism changes. In

5 a changing environment genetic variation is beneficial, increasing the likelihood of at least some offspring being better adapted to the environment and hence raising the probability of the parents’ genes being passed on one generation further. Both the sexual mixing of genetic information and random mutations are involved in creating this variation. Obviously the variation is good only for the individuals receiving the best variations and the highest fitness. The genes are selfish. The vertebrates, or animals with backbones, comprise neither the largest nor most diverse group of animals. Nevertheless, this group is by far the most investigated among all animals for several reasons. We ourselves being members of the vertebrate phylum is obviously one. Another is that vertebrates are comprised of many hard parts and have left relics throughout evolution yielding an enormous fossil record which has made them particularly useful for reconstructing evolutionary processes (Benton, 1990; Kardong, 1994). The divergence of vertebrates is thought to have started at least 500 million years ago (Benton, 1993) in the Cambrian era. The ancestor to all vertebrates possibly resembled the lancelet, Amphioxus, a small tadpole-like segmented organism with a notochord and an anterior assembly of neurons forming a simple brain (Benton, 1990; Shimeld & Holland, 2000). A number of large alterations to the genome of this postulated ancestor may possibly have created the prerequisite for the enormous radiation resulting in mammals, birds, reptiles, amphibians and fishes (Meyer & Schartl, 1999; Shimeld & Holland, 2000). In order to gain a greater understanding of the evolution of the gene family studied in this thesis, the NPY receptors, we selected species from key lineages within the vertebrate tree from which to clone and characterize the genes. As the NPY receptors in mammals have been extensively studied, the species we chose to study represent groups that separated from the vertebrate tree at more or less evenly spaced intervals prior to the origin of mammals. With this approach we hoped to gain insights into the processes that have shaped the gene family in this large and diverse phylum. Below follows short presentations of each of the species studied.

Birds Class: Aves Birds evolved from diapsid reptiles in the middle Jurassic period, and are sometimes grouped together with reptiles in one taxonomic category. Reptiles and birds have similar bone and muscle structures as well as shell- encased eggs. Basic avian design has proven to be very adaptable resulting

6 Pr c ot Fishes Tetrapods h o o - rda t Agnatha Gnathostomata e MYA s

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Fig 1. Vertebrate evolution. The vertical scale on the left represents time in millions of years before present. Bars in the graph represent the extant groups of vertebrates as well as the protochordate class cephalochordates of which Amphioxus is a member. Myxinii and Cephalaspidomorphi (e.g. lampreys) constitute the agnathan or jawless fishes. The Chondrichthyes or cartilaginous fishes are comprised of the Elasmobranchii (e.g. sharks) and Holocephali. The Osteiichthyes or bony fishes are represented by two subclasses. The Actinopterygians are comprised of the Chondrostei (e.g. sturgeons) and Neopterygia within which the teleosts (e.g. zebrafish and cod) are found. The Sarcopterygians (Dipnoi and Crossopterygians) are the second subclass. The broad groups of chordates indicated at the top of the graph include agntha and gnathostomes (jawless fish and jawed species respectively), and fishes and tetrapods (four-limbed species) which encompass the classes below them. Thick arrows indicate postulated genome duplications. A thinner arrow indicates the assumed position for the duplication event that resulted in PP. Modified from (Kardong, 1994) and (Benton, 1993).

7 in an extensive radiation and diversification. Birds are today the second largest class of vertebrates in terms of number of species, superceded only by the bony fishes. Unique traits for the avian class are feathers and toothless beaks. One of the most well known fossils is Archaeopteryx, which very well represents an ancient intermediate between reptiles and birds having both teeth and a beak as well as feathers. The fossil specimens support monophyly of reptiles and birds (Gauthier, 1986) and exhibits several anatomical traits pointing to Archaeopteryx being able to fly in a similar fashion to modern birds (Feduccia & Tordoff, 1979). The domestic chicken (Gallus gallus) has become the model organism of choice for research in this class of vertebrates, greatly due to its commercial value and the fact that it can easily be kept and bred. Within the field of developmental biology the chicken has since long been among the most investigated species (Cheng, 1997; Dupin et al., 1998).

Advanced bony fishes Class: Actinopterygii, Subclass; Neopterygii, Division: Teleostei The actinopterygians are the ray-finned fish that together with sarcopterygians (i.e. lungfish and coelacanths) comprise the bony fishes. Actinopterygii are divided into three groups: chondrosteans, holosteans and teleosts (Nelson, 1994). Holosteans and teleosts are referred to as the neopterygians or advanced ray finned fish. The most advanced of the bony fishes are the teleosts which literally means “terminal” and “bone”, reflecting on their advanced anatomy. This is the most diverse group of all vertebrates encompassing more than 20,000 species with an extremely wide geographical distribution. Despite this extreme diversity and divergence within the group some common characteristics are observed that form the basis for their classification, among others heterocercal tails, swim bladders and circular scales. The commercially important species of fish such as cod (Gadus morhua), salmon (Salmo spec.), and herring (Clupea harengus) are all teleosts. The zebrafish (Danio rerio) is a tropical freshwater species. The zebrafish has during the past decade become the favored model species for developmental and molecular biologists, not because of any direct commercial value but because of its advantages as a laboratory animal (Fjose, 1994; Wixon, 2000). Due to the considerable amount of investigation done into the genetics of the zebrafish, detailed linkage maps and information concerning the teleost genome are available (Amemiya et al., 1999; Postlethwait et al., 1999; Postlethwait et al., 2000). The

8 zebrafish also has become a favored model organism for studying vertebrate development including the nervous system (Blader & Strahle, 2000).

Sharks Class: Chondrichthyes, Subclass: Elasmobranchii, Superorder: Euselachii The extant chondrichthyan or cartilaginous fishes consist of two groups, the sharks and rays (Elasmobranchii) and chimaeras (Holocephali) which appear to have split from one another soon after the appearance of the first cartilaginous fishes. Both groups are characterized by cartilaginous skeletons, placoid scales and pelvic claspers (Maisey, 1986). The distinctive placoid scales appear in the fossil record in the late Silurian era (Zangerl, 1981) approximately 420-430 MYA, indicating that the chondrichthyans arose at this point, although later periods of radiation resulted in the modern suborders. The cartilaginous skeletons appear to have resulted from a secondary loss as fossils indicate that bone tissue was present in the skeletons of chondrichthyan ancestors. Also, traces of bone tissue are present in modern sharks (Hall, 1982). Like all fish the chondrichthyans are denser than water, but lack the swim bladder present in most bony fishes. This is compensated by an unusually fatty liver that increases buoyancy. Also, unlike most bony fishes, chondrichthyans tend to produce relatively small numbers of young and some species even retain their young in the reproductive tract until fully developed (Kardong, 1994). The phylogeny of elasmobranchs has been difficult to resolve, and present literature is far from a consensus on the matter (Compagno, 1973; Janvier, 1996; Nelson, 1994). Primarily, it is not agreed upon whether rays and skates form a monophyletic group with sharks (Compagno, 1977). However, the modern sharks appear to consist of at least eight orders which primarily diverged during the mid to late Jurassic period, some 150-200 MYA (Shirai, 1996), separating the major groups of sharks by a time span equivalent to that of reptiles and birds. Within the order Squaliformes (Nelson, 1994) is the family Squalidae or dogfish sharks. The most common species found in both the northern and southern hemispheres is the spiny dogfish, Squalus acanthias. Although the spiny dogfish is similar in name and appearance to the common dogfish Scyliorhinus canicula, a shark species upon which research in the NPY field has been performed (Alvarez- Otero et al., 1995; Conlon et al., 1991a), several hundred million years of evolution separate the two species (Shirai, 1996).

9 Fig 2. The spiny dogfish, Squalus acanthias.

Lampreys Superclass: Agnatha, Class: Cephalspidomorphi, Order: Petromyzonti- formes Agnathan means literally “without jaw”. Two classes of living agnathans exist, the hagfish (Myxinii) and lampreys (Petromyzontiformes), while numerous extinct species are known by their fossils. The agnathan fishes differ from other vertebrates by lacking bone tissue and a biting apparatus derived from branchial arches. This initially led to the two classes of agnathans to be grouped together, although this has been disputed. Bone tissue first appeared in extinct agnathans, or ostracoderms (lat. “bone” and “skin”) in the late Cambrian, more than 440 MYA. It is now believed that the lack of bone tissue in hagfish is a primary trait, while the lampreys lost bone tissue secondarily. Several other physiological and anatomical traits support the lampreys being more closely related to all other vertebrates than the hagfish (Janvier, 1981; Janvier, 1986; Maisey, 1986). Indeed, if the hagfish lacks bone as a primary trait, and thereby vertebrae, they cannot be considered vertebrates but instead chordates. Recently, some sequence data indicated that all extant agnathans form a monophyletic group (Kuraku et al., 1999; Mallatt & Sullivan, 1998; Suga et al., 1999) while other molecular data sets support the view that hagfish and lampreys indeed represent separate clades (Gursoy et al., 2000). This illustrates the difficulty in determining the relationship of these two classes with each other and the gnathostomes (lat. “jaw” and “mouth”) or all other vertebrates (Delarbre et al., 2000). While the exact relationship with the hagfish is not completely clear with respect to molecular data, it appears quite certain that at least one large duplication event in the genome of jawed vertebrates separates gnathostomes from lampreys (Freitag et al., 1999; Sharman et al., 1997;

10 Stock & Whitt, 1992). Unique duplications of certain genes in the lamprey genome emphasize the differences between the two groups (Takahashi et al., 1995; Wang et al., 1999).

Fig. 3. The European river lamprey, Lampetra fluviatilis.

MOLECULAR EVOLUTION

Molecular evolution includes two major areas of study. The first addresses the causes and consequences of evolutionary changes to molecules. The second area of research uses this as a tool to reconstruct the evolutionary history of organisms (Li, 1997). This has become an increasingly interesting field largely because of the enormous technical advances in recent years providing ever-greater data sets. During the past few years the entire genomes of several organisms have been sequenced, including yeast (Saccharomyces cerevisiae) (Consortium, 1997), a nematode (Caenhoradbitis elegans) (Consortium, 1998) and the fruit fly (Drosophila melanogaster) (Adams et al., 2000). The sequence of the human genome has recently been completed and has become publicly accessible (Lander et al., 2001). The work presented in this thesis encompasses both of the fields mentioned above. We have in part studied a group of molecules involved in neuronal communication and how they have changed over the course of vertebrate history. Also our studies aim to aid in the reconstruction of vertebrate evolution. It is apparent that data from different fields of science must be incorporated in order arrive at the true order of events and to avoid logical errors. Molecular information alone is not sufficient to resolve interspecies relationships but is an excellent tool to trace the actual changes to individual molecules during evolution. Paleontology, morphology and anatomy, embryology as well as physiology are all

11 important fields of study involved in elucidating evolution. Molecular methods have not replaced but complemented these areas of research. Mutations within DNA sequences are what generate variation. This variation is subjected to selection which results in evolutionary change. Each time a mutation occurs that does not decrease fitness, it can become fixed in the genome and become part of the evolutionary record. Since mutations occur at random, they will gradually accumulate over time. The longer the time since two species separated the more extensive the differences compared to the genome of their common ancestor, as well as to each other. By comparing the genomes of several species the ancestral sequence can be stipulated making the reconstruction of molecular evolutionary possible. The rate at which mutations occur can vary between lineages and species as well as between genes for several reasons. If the environmental constraints upon a species are constant, variation is not beneficial and mutations will most often result in lower fitness. This would be reflected by fewer mutations accumulated over a certain period of time. If environmental factors favor variation the number of accumulated mutations over the same period of time would appear larger. Differing rates of evolution of genes depend upon the functional constraints on the gene product. It can be difficult to estimate the true number of mutations when studying sequences separated by large time spans, having accumulated large numbers of mutations. A very large number of mutations can also result in re-mutations, reverting a mutation back to resemble the sequence of the ancestor, disguising the first mutation. Also, the constraints upon genes coding for proteins vary depending on their physiological importance, with those absolutely vital tolerating change to a lesser degree than others.

GENE DUPLICATIONS

Mutation is not the sole mechanism by which evolution proceeds. The increasing amount of sequence information has revealed that genes can be grouped into families and superfamilies on the basis of sequence similarity. The most reasonable explanation for two sequences resembling each other is that they evolved by duplication of a common ancestor. The process of gene duplication is an important mechanism of evolutionary change. If a gene exists in two copies, due to a duplication of that particular genomic segment, it will suffice that one of the copies remains constant allowing the second to accumulate mutations and thereby allowing the gene product to acquire novel properties and functions. Another possibility is that the two

12 daughter genes will specialize on different aspects of the mother gene’s functional repertoire. Alternatively one of the copies could accumulate so many mutations that the function of the gene product eventually would be lost (Force et al., 1999; Nadeau & Sankoff, 1997), resulting in a pseudogene, with no functional constraints whatsoever. Gene duplication can occur on different scales. Local duplication of chromosomal segments occurs during crossing over events in meiosis will result in two gene copies. Duplication of entire chromosomes or even entire genomes result in copies of a multitude of genes (Ohno, 1970). This could possibly facilitating leaps in evolutionary processes, allowing duplicates to acquire alternate or specialized functions (Ganfornina & Sanchez, 1999; Wagner, 1998). It has been postulated that at least two large-scale duplications have taken place during the evolution of vertebrates (Holland, 1999; Lundin, 1993; Sidow, 1996). In mammals it has been known for some time that many genes exist in groups of four similar copies. Among these are the HOX gene clusters that are involved in embryonic development. It has been observed in several cases that the gene quartets consist of two pairs, implying again that a first duplication event resulted in two genes that were subsequently duplicated in a second event. This would then have resulted in two pairs of genes sharing a single common ancestor. The large-scale duplications in vertebrate evolution have been supported by investigation of other gene families (Kasahara, 1998; Katsanis et al., 1996; Lundin, 1993). The increased anatomical complexity of vertebrates could possibly be correlated to an increased genetic complexity as a result of the postulated genome duplications (Holland et al., 1994; Shimeld & Holland, 2000). Another notable trait of gene duplicates is that they are often located to paralogous chromosomal segments. If an entire chromosome is duplicated, all genes located on one will have duplicates appearing in the same order on the second (Lundin, 1993; Pebusque et al., 1998). If genes A, B and C are located in that order on a certain chromosomal segment which is subjected to a large-scale duplication, the two resulting daughter chromosomal segments will contain genes A1, B1, C1 and A2, B2, and C2 respectively. A subsequent duplication of both daughter segments would then result in two pairs of chromosomal segments, the first pair harboring the genes A1a, B1a, C1a and A1b, B1b, C1b and the second pair with A2a, B2a, C2a and A2b, B2b, C2b. Chromosomal localization of gene families in

13 pig, rat, cow and zebrafish have indeed revealed a respectable number of paralogous chromosomes and chromosomal segments in each of these species (Andersson et al., 1996; Aparicio, 1998; O'Brien et al., 1999) (Fig. 4).

AD R NPYR

Local duplications

FGFR DR D ADR: A1 B A2 GABR A SN C EGR ANX NPYR NPYR NPYR GRIA GLRA A Large-scale duplications

HSA4 FGFR3 DR D 5 ADR: A2C GABR A2 SN CA ANXA5NPY5RNPY1R NPY2R GRIA2 GLR A3

HSA5 FGFR4 DR D 1 ADR: A1B B2 GABR A1/6 SN CB EGR 1 ANXA6 NPYR NPY6R NPYR GRIA1 GLR A1

HSA10 FGFR2 ADR: B1 A2A SN CG EGR 2 ANXA7 NPYR NPY4R NPYR

FGFR1 ADR: A1C B3 A2B ANXA7 NPYRYb? NPYR

HSA8 HSA2 HSA? Fig 4. The proposed evolution of paraloguous chromosomal segments, harboring the NPY receptor genes and other gene families. ADR, adrenoreceptor; GRIA, glutamate receptor; GABAR, GABA receptor; FGFR, fibroblast growth factor receptor.

NEUROSCIENCE AND G-PROTEIN COUPLED RECEPTORS

The brain is the organ that obtains information about the environment, processes and stores this information, generates behavior and in humans is responsible for such aspects as emotions, abstract thoughts and our perception of the world and ourselves. In other words the brain makes us what we are. It could even be argued that the function of all other organs is to support the brain. For these reasons it is of great interest to study and attempt to understand the processes of the brain and nervous system. In the age of molecular biology we now own the means to obtain a greater understanding of these processes, and the processes of disease and pathology affecting them. The human brain is comprised of approximately one hundred thousand million neural cells, organized in networks. Communication within the nervous system occurs by means of both electrical and chemical signaling. Each cell can have up to 10,000

14 connections with other cells by means of chemical synapses. The electrical signals conveyed by a cell influence emission of chemical substances that cause responses by adjacent cells. These transmitter substances are either modified amino acids or short chains of amino acid residues, peptides, such as neuropeptide Y. The transmitter substances emitted by a cell are received by receptor molecules on both other neurons and on the signaling neuron. This chemical signal is then converted in to an electrical signal in the receiving neuron. The receptors on the emitting neuron also receive the chemical signal and can by feedback regulate the emission of transmitters. The large array of transmitter substances in combination with an even larger number of specific receptors lay the ground for the great complexity of the nervous system. The largest groups of structurally related receptors are the G-protein coupled receptors (GPCR). Genes that code for these receptors have been estimated to comprise approximately 1% of the mammalian genome, numbering between 1000-2000 genes (Bockaert & Pin, 1999). The receptors are characterized by seven transmembrane _-helices spanning the cell membrane and connected by loops on the extra- and intracellular sides of the membrane, hence the alternative names seven-transmembrane (7TM) or heptahelix receptors (Fig. 5) (Baldwin, 1993; Baldwin, 1994; Ji et al., 1998). GPCRs bind synaptic transmitters such as monoamines and peptides. Numerous GPCRs are involved in light perception, the opsine receptors and many GPCRs are odorant receptors involved in olfaction. The intracellular portions of the receptors activate G-proteins (Guanine binding nucleotide proteins) when activated by a ligand. These proteins are trimers and numerous variants of the subunits provide the complexity for G-protein signaling (Hamm, 1998). The G-proteins either stimulate (Gs) or inhibit (Gi) adenylyl cyclase and thus the synthesis of cAMP. The activated G- proteins also influence intracellular signaling systems such as cGMP, phopholipases, phoshodiesterases or ion channels, relaying the extracellular signal to an internal chemical response. The broad functional repertoire of the GPCR superfamily suggests ancient evolutionary origins, and they are indeed found in such distant lineages as plants, yeasts and slime molds (Josefsson, 1999; Josefsson & Rask, 1997).

15 NH2

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I II COOH III Fig. 5. Schematic diagram of G-protein coupled receptor (GPCR) in a cell membrane. The amino terminal is located on the extracellular side of the membrane. Seven _-helices span the membrane, creating a pocket for ligand binding. The carboxy terminus, located on the intracellular side, binds the G-protein complex (not shown). On the upper right is a flattened two-dimensional diagram of a GPCR in a cell membrane.

NPY PEPTIDE FAMILY

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide form a family of structurally related neuroendocrine peptides. All peptides are 36 amino acids long, with the exception of chicken PYY which is one amino acid longer, and share a hairpin-like structure called the PP-fold (Fuhlendorff et al., 1990). The three-dimensional structure has been deduced both by NMR (Boulanger et al., 1995; Saudek & Pelton, 1990) and X-ray crystallography (Blundell et al., 1981). The first eight amino acids form a proline type II helix and residues 15-32 form an a-helix. NPY is one of the most conserved peptides known. Out of 36 residues, only 2 vary among mammals, and 14 among vertebrates. PYY evolves at a somewhat faster rate with eight positions varying within the mammalian lineage. PP

16 evolves most rapidly of all members of the peptide family (Larhammar, 1996; Larhammar et al., 1998). Apart from these three peptides two further members have been isolated from certain species. PY is found only in the acanthomorph fishes, a subclass of the teleosts, and probably resulted from a gene duplication of the PYY gene within this lineage (Cerda-Reverter & Larhammar, 2000; Cerda-Reverter et al., 1998). Peptide MY (PMY) found in lamprey, an agnathan fish, most likely arose due to a duplication of the ancestral PYY gene as well (Conlon et al., 1991b; Söderberg et al., 1994; Wang et al., 1999). NPY has been found in all vertebrate lineages and is extremely well conserved (Cerda-Reverter & Larhammar, 2000). A peptide with a high degree of identity with and effects similar to NPY has been isolated from the snail, Lymnea stagnalis (Tensen et al., 1998). Moreover, the insect peptide NPF (Leung et al., 1992) is possibly related to the NPY peptides illustrating the vast span of evolution over which NPY has survived, suggesting vital functions. Another peptide that seems to be a close relative of PYY is seminalplasmin, isolated from bovine semen (Herzog et al., 1995). Although the gene for seminalplasmin is nearly identical to the PYY gene, a frameshift mutation makes the amino acid sequence quite different.

NPY NPY (Tatemoto, 1982a; Tatemoto, 1982b) has several functions of which some of the most pronounced are induction of feeding (Clark et al., 1984; Dube et al., 1994; Levine & Morley, 1984; Lopez-Patino et al., 1999; Nakajima et al., 1994), thermoregulation, regulation of circadian rhythm, reproduction and vasoconstriction. NPY is extremely abundant in the CNS as well as widespread in the PNS, co-localized with noradrenalin (NA) in sympathetic nerves. In the hypothalamus NPY neurons project from the arcuate nucleus to the paraventricular nucleus, which is the primary site for integration of energy balance (Leibowitz et al., 1988). NPY enhances the vasoconstriction mediated by NA (Dumont et al., 1992; Franco-Cereceda & Liska, 1998). Other effects of the peptide are regulation of circadian rhythms (Albers & Ferris, 1984), mood and memory (Heilig & Widerlov, 1990; Wahlestedt et al., 1993). Several pathological conditions have been associated with changes in NPY function including anorexia, bulimia neurosa, diabetes as well as several cardiovascular diseases (Hulting et al., 1990; Ullman et al., 1990). Knockout experiments of NPY in mouse were reported to result in no large phenotypic alterations with respect to food intake or obesity (Erickson et al., 1996a; Erickson et al., 1996b), although

17 some effects in weight regulation have been seen in leptin and NPY deficient mice (Erickson et al., 1996b). More recently, independent studies of these mice did revealed reduced food-intake and growth (Bannon et al., 2000).

PYY and PMY PYY (Tatemoto & Mutt, 1980) is released from the gastrointestinal tract in response to food intake as well as being located to certain populations of neuronal cells in the CNS and PNS. Apart from the functions of PYY in the gut, inhibiting gall bladder and pancreas secretion and gut motility (Hazelwood, 1993; Tatemoto, 1982a), it could also have neuronal functions (Jazin et al., 1993). Like NPY, PYY has been found in neurons of several lower vertebrates as well as all investigated gnathostomes (Cerda-Reverter & Larhammar, 2000; Cerda-Reverter et al., 1998; Söderberg et al., 1994). In the lamprey, Lampetra fluviatilis, one of the species studied in this thesis, a duplication of the PYY gene appears to have resulted in two similar peptides called PMY (Wang et al., 1999) and PYY. PMY has so far been found in several species of distantly related lampreys, including Geotria australis and Petromyzon marinus (Conlon et al., 1991b), suggesting this duplication occurred early in the lamprey lineage. In Petromyzon, PMY has been detected in the intestine and appears to play a role in the regulation of estradiol and maturation in females (Conlon et al., 1994). PYY has been shown to bind the same receptor subtypes with similar affinities as NPY and mimics its effects, which raises questions as to whether or not the two peptides can overtake one another’s functions if the other is lost. This could then provide a possible, albeit somewhat speculative explanation for the mild phenotype of NPY knockout mice. However, no compensatory expression of PYY has been reported in these KO mice.

PP It seems as if PP (Kimmel et al., 1975; Kimmel et al., 1968) is the most recent addition to the NPY peptide family, most likely having arisen in the tetrapod lineage after the split from piscine lineages. The PP and PYY genes in human are located near each other on chromosome 17 (Hort et al., 1995). This suggests that PP arose due to a local duplication, not a genome or large-scale duplication, explaining why it has not been found in any non- tetrapod. PP was the first of the NPY peptide family to be isolated and is almost exclusively expressed in the endocrine pancreas. Plasma levels of PP increase in response to food intake and influence excretion of insulin

18 (Hazelwood, 1993; Schwartz, 1983). PP is the most rapidly evolving peptide in the family exhibiting only seven constant residues when comparing among tetrapods (Cerda-Reverter & Larhammar, 2000).

NPY RECEPTORS

The NPY receptors form a family within the G-protein coupled receptor superfamily. The mechanisms of G-protein activation by the NPY receptors are still incompletely known although inhibition of cAMP and elevation of intracellular calcium appear to be important (Herzog et al., 1992; Larhammar et al., 1992; Michel et al., 1998). In mammals five separate Y receptor subtypes have been identified. All of these subtypes have been cloned in mouse, human (Michel et al., 1998), pig (Wraith et al., 2000) and guinea pig (Berglund et al., 1999; Eriksson et al., 1998; Lundell et al., 2001; Sharma et al., 1998; Starbäck et al., 2000). Although pharmacological data has indicated the existence of an additional subtype in mammals, referred to as Y3, no such receptor has yet been found (Michel et al., 1998). Subtypes have been isolated in other tetrapod species such as chicken Y2 (Paper I) and frog Y1 (Blomqvist et al., 1995) as well as from several non-tetrapod vertebrates. Three receptors have been isolated from the teleost zebrafish (paper IV) (Ringvall et al., 1997; Starbäck et al., 1999) and one from cod (Arvidsson et al., 1998) (Fig. 6). Recently additional receptors from species even more distant to mammals have been cloned, including three from the shark Squalus acanthias (paper III) and one from the agnathan lamprey, Lampetra fluviatilis (paper II). Outside the vertebrate lineage a NPY receptor and ligand have been identified from Lymnea stagnalis (Tensen et al., 1998) as well as proposed NPY receptors from Drosophila melanogaster (Li et al., 1992) and Caenorhabditis elegans (de Bono & Bargmann, 1998). However as no ligands have been identified in these two species, the definitive membership in the Y receptor family is uncertain. Alignments and phylogenetic analysis of the NPY receptors reveals a low degree of identity for several subtypes, as little as 30%, which is near the degree of identity seen between families of GPCRs. This suggests ancient origins of the NPY peptide-receptor system. The subtypes found in mammals possibly originated by means of gene duplication prior to the origin of vertebrates (Larhammar et al., 2001). Within the NPY receptor family, subtypes Y1, Y4 and y6 along with the three subtypes only found in teleosts exhibit higher levels of identity to each other (50%) than to either

19 Y1 Human Y1 Pig Y1 Dog Y1 Rat Y1 Mouse Y1 Guinea Pig Y1 Chicken Y1 Xenopus Y6 Mouse y6 Rabbit Y1 Y4 Human subfamily Y4 Pig Y4 Guinea Pig Y4 Rat Y4 Mouse Yb Zebrafish Yc Zebrafish Yb Cod Ya Zebrafish Y5 Human Y5 Guinea Pig Y5 Dog Y5 Y5 Rat Y5 Mouse Y2 Human Y2 Bovine Y2 Rat Y2 Mouse Y2 Y2 Guinea Pig Y2 Pig Y2 Chicken

Fig 6. Neighbor-joining distance tree of Y receptor amino acid sequences generated with PAUP software. Alignments were made using PAM 250 and BLOSSUM weighting matrices. The divergent cytoplasmatic and extracellular regions were excluded from the analysis. Arrows on the right indicate Y1 subfamily, Y2 group and Y5 group. Human bradykinin receptors B1 and B2 were used as an outgroup. The tree is unrooted.

Y2 or Y5 (30% identity) forming the Y1 subfamily (Fig 2). Y2 and Y5 are also only 30% identical to each other. Y1, Y2 and Y5 have been localized to the same chromosomal segment in human, while Y4 and y6 have been mapped to separate chromosomes. An attractive explanation for evolution of Y receptors can be inferred from the theory of multiple vertebrate genome duplications (Fig 3). It appears possible that the original chromosome containing the genes coding for the three most divergent subtypes Y1, Y2 and Y5 was duplicated twice early in vertebrate evolution

20 resulting in three copies of at least the Y1 ancestor. No genes constituting putative duplicates of Y2 or Y5 have been found as of yet, but could be expected to be found in the proximity of the Y4 or y6 genes, or on a yet another chromosome. It appears that after the large-scale duplication events a considerable number if duplicates become redundant, eventually mutating to pseudogenes, and further beyond recognition (Nadeau & Sankoff, 1997; Wagner, 1998). In mammals NPY receptor are expressed widely and the expression patterns overlap to a large extent (Dumont et al., 1998). Y1 and Y5 seem to be co-localized in the brain of the rat (Parker & Herzog, 1999). The distribution of receptors appears to differ between mammalian species. Y1 is the most abundant receptor in the rat brain (Gehlert et al., 1992) while Y2 is predominant in the human brain (Gehlert et al., 1996; Jacques et al., 1997; Jacques et al., 1996) as detected by Northern hybridization.

Y1 The first NPY receptor was cloned from the rat (Eva et al., 1990) and subsequently found to represent the Y1 subtype (Herzog et al., 1992; Larhammar et al., 1992). It has now been cloned in a number of mammals and other tetrapods (Larhammar et al., 1998) as well as from the shark (paper III). The Y1 gene is highly conserved showing 92-95% amino acid identity between mammals. Y1 appears to be the most important mediator of the vasoconstriction effects of NPY (Grundemar et al., 1992; Malmstrom et al., 1998) as well as being involved together with Y5 in regulation of feeding (Mikkelsen & Larsen, 1992; Schaffhauser et al., 1997; Schaffhauser et al., 1998). The Y1 and Y5 genes, located on HSA4, appear to possibly have co-ordinated transcription (Herzog et al., 1997). All known Y1 sequences contain an intron after the fifth TM region and is the only NPY receptor gene found to contain an intron in the coding sequence. In mammals the intron is very short, measuring only 100-120 bp, but seems to be expanded in the shark (paper III). The mammalian Y1 receptors bind NPY and PYY with roughly equal affinity and PP with lower affinity (Berglund et al., 1999).

Y2 Y2 was first cloned in rat is also highly conserved showing ca 90% identity at the amino acid level between mammalian orders and 75-80% between mammals and chicken (paper I), but shows only 30% identity to other Y receptor subtypes. Y2 is the most abundant NPY receptor in the human

21 Ancestral Y receptor

Y2 ancestorY1/5 ancestor

Y2 ancestor Y1 subfamily Y5 ancestor I ancestor

II Y2Y1/6 Y5

Y4/b

HSA4 III Y2 Y1 Y5

HSA5 Y6

HSA10 Y4

Fig 7. A possible scheme for the duplication of NPY receptor genes in vertebrate evolution. I. Local duplications of an ancestral NPY receptor gene resulted in a chromosomal segment containing three receptor genes. This is the expected arrangement in cephalochordates. II. A genome duplication resulted in two copies of the original chromosomal segment. This could possibly be similar to the arrangement in agnathan fishes. III A second genome duplication resulted in four copies of the original segment. This is in agreement with the arrangement in human. Human chromosome numbers are indicated on the left. The insertion of an intron in the Y1 gene is indicated by a black box. brain with the highest concentrations found in the hippocampus suggesting it cold be involved in memory (Flood et al., 1989) and effects circadian rhythms (Huhman et al., 1996). In the gastrointestinal tract Y2 is involved in regulation of secretion (Cox & Cuthbert, 1990). It also plays an important role in vasoconstriction (Malmstrom et al., 1998) and angiogenesis (Zukowska-Grojec et al., 1998). Y2 is also found presynaptically where it acts as a regulator of NPY transmission (Wahlestedt & Håkanson, 1986). Recently Y2 has been found to be involved in regulation of energy balance as well (Naveilhan et al., 1999).

22 NPY and PYY bind mammalian Y2 with approximately equal affinities but PP has no detectable affinity for Y2 (Gehlert et al., 1996; Gerald et al., 1995). The chicken Y2 receptor has a somewhat different pharmacological profile (paper I).

Y4 In mammals the Y4 gene has an accelerated rate of evolution compared to that of Y1 and Y2 making it the receptor that diverges most between orders of mammals (Larhammar et al., 1998). The function of Y4 is not yet completely clear, but evidence exists for functions within the gastrointestinal tract in rabbit (Feletou et al., 1999). PP binds Y4 with higher affinity than NPY or PYY, which is why Y4 is sometimes referred to as PP1 (Bard et al., 1995; Lundell et al., 1995). Notably PP exhibits a relatively high rate of evolution as well. As PP is found primarily in the gastrointestinal tract, its seems reasonable that Y4 should be involved in gastrointestinal functions, although evidence exists for Y4 also being expressed in the CNS (Parker & Herzog, 1999). Considering that PP only exists in tetrapods, the function and pharmacology of Y4 receptors in non- tetrapod gnathostomes will most likely differ substantially. Y4 in Squalus (paper III) represents the first non-tetrapod Y4 to be cloned and should contribute with important insights into the origin and evolution of Y4.

Y5 The Y5 receptor attracted considerable attention after it was cloned (Gerald et al., 1996), mainly because its stipulated role as a regulator of feeding (Criscione et al., 1998; Schaffhauser et al., 1997). Y5 shows an equal degree of identity to Y2 as to the Y1 subfamily subtypes, i.e. approximately 30%. It is longer than the other Y receptors due to an additional 100 amino acid residues in the third intracellular loop. Phylogenetic analyses and chromosomal mapping indicate that Y5 and Y1 appeared as a results of the second local gene duplication prior to vertebrate divergence, since the Y2 subtypes branches basal all other Y receptors. NPY and PYY show equal affinity for the Y5 receptor (Larhammar et al., 1998). Also PP binds to Y5 with an affinity that may be in the physiological range, but it is unclear if PP may reach the sites of Y5 in vivo.

23 y6 The y6 subtype was the most recent Y receptor to be cloned in mammals (Gregor et al., 1996b; Weinberg et al., 1996). The rate of evolution of y6 is also greatly accelerated in the mammalian lineage as compared to most other GPCRs with only 78-81% identity between mammalian orders. This appears to be due to a redundancy of the subtype in these species. In humans as well as other primates (Gregor et al., 1996a) and pig (Wraith et al., 2000) the gene has frame-shift mutations rendering it a pseudogene. In the rat the y6 gene appears to have been lost (Burkhoff et al., 1998). In mouse, rabbit and peccary, from which y6 has also been cloned the reading frame is intact but no physiological correlate has been found, why the receptor is designated with a lower case y in the receptor nomenclature (Michel et al., 1998). Due to the fact that the y6 sequence still is intact in several species it does seem likely that the redundancy has arisen recently in evolution and could possibly be a unique trait in certain orders of mammals. Preliminary data from the marsupial wallaby (Salaneck unpublished) and the chicken (Fredriksson, Salaneck & Larhammar, unpublished) support this notion as their y6 genes appear to have functional status. A likely functional Y6 gene has also been isolated from the shark (paper III), where it seems to exhibits a more moderate rate of evolution. The great sequence differences between y6 in mammals correlates with the considerable differences in receptor pharmacology for the mouse and rabbit receptors, leaving many questions about the functional role of y6 unanswered. The Y6 clones from chicken and shark will hopefully resolve some of these questions as well as allow studies of anatomical distribution and pharmacological characterization.

Ya and Yb/c The three receptors from zebrafish, Ya (Starbäck et al., 1999), Yb (paper IV) and Yc (Ringvall et al., 1997), along with Yb from cod (Arvidsson et al., 1998) all show a high degree of identity with the Y1 subfamily sequences, but do not appear to be orthologues to any of the known mammalian subtypes. Despite considerable efforts, no orthologues of these receptors have been found in mammals. Likewise, no orthologues of Y1, Y4 or y6 have been found in teleosts. A possible complication in identifying teleost orthologues is the additional genome duplication shown to have taken place in this lineage (Amores et al., 1998; Postlethwait et al., 2000). The appearance of multiple duplicates most likely led to the duplicate genes acquiring novel or specialized functions compared to those of the pro-

24 orthologue. Also, an extensive number of the gene duplicates arising from this event seem to have been subsequently lost as seen from investigation of other gene families in zebrafish (Postlethwait et al., 2000). The zebrafish Yb and Yc genes appear to have resulted from a local duplication in the zebrafish lineage, as they are both equally identical to Yb in cod. The position of Ya in phylogenetic trees has been somewhat more confusing. The sequence of Ya is more similar to Yb/c and Y4 than either Y1 or y6. The pharmacological properties of the Yb and Yc receptors resembles that of Y1, while Ya displays a strikingly indiscriminate binding profile (Berglund et al., 2000). The anatomical distribution of the teleost receptor mRNAs have been difficult to determine, possibly due to low levels of transcription. Yb has been detected in eye and intestine by RT-PCR (paper IV).

NPY receptors in invertebrates Several Y receptor candidates have been isolated from non-mammalian as well as invertebrate species. The most convincing of the invertebrate candidates is that cloned from the snail Lymnea stagnalis, from which an NPY-like peptide has also been isolated and found to bind to and influence intracellular Ca++ -levels (Tensen et al., 1998). In the fruit fly Drosophila melanogaster a receptor with a certain degree of identity to mammalian subtypes was shown to bind mammalian NPY and PYY (Li et al., 1992), but no endogenous ligand has been found. A third invertebrate receptor has been isolated from the nematode Caenorhabditis elegans. This sequence also exhibits similarities to mammalian NPY receptors and seems to be involved in feeding behavior (de Bono & Bargmann, 1998).

25 RESEARCH AIMS

The overall aim of this doctoral work was to investigate the evolution of the NPY receptor family in the vertebrates. At the start of this project, evidence had been presented that the vertebrate genome has undergone chromosomal duplications or even tetraploidizations (Holland et al., 1994; Lundin, 1993; Ohno, 1970; Ohno, 1973). During the course of this work, additional evidence has accumulated that supports this scenario (Holland, 1999; Lundin, 1999; Meyer & Schartl, 1999; Postlethwait et al., 2000). However, conflicting views are maintained by some scientists who argue that parology groups may have arisen by independent parallel aggregation of gene clusters (Hughes, 1999; Skrabanek & Wolfe, 1998; Wang & Gu, 2000). The NPY receptor genes described in this thesis have been evaluated with regard to these alternative hypotheses and clearly support the chromosomal duplication/tetraploidization alternative. The availability of endogenous ligands from the vertebrate species investigated facilitated pharmacological characterization of the novel receptors. Taken together, this information improves our understanding of the basic mechanisms by which the vertebrate genome has evolved, and provides information concerning the co-evolution of this particular family of peptides and receptors involved in important physiological and neurological processes.

26 PAPER SUMMARIES: RESULTS AND CONCLUSIONS

PAPER I Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2 In order to gain a greater understanding of the evolution of the NPY receptors we undertook the cloning and characterization of the Y2 receptor in the chicken Gallus gallus. A genomic clone was isolated by low- stringency screening of a genomic phage library with a combination of rat and human Y2 probes. The chicken Y2 receptor sequence exhibits a 75- 80% identity with mammalian subtypes, which is agreement with the Y2 subtypes evolving at a relatively slow rate compared to other NPY receptor subtypes. A higher degree of identity with mammalian receptors was seen within the transmembrane regions while the cytoplasmatic tail was surprisingly divergent. Expression of the receptor in a stable cell line showed that the receptor did not bind the mammalian Y2 selective antagonist BIIE0246, but did bind the modified peptide p[Leu31,Pro34]NPY. Interestingly, this modified peptide rendered more similar to PP, the primary ligand for Y4, has not been shown to bind to mammalian Y2. A plausible explanation for this is that chicken PP differs from mammalian PP by having a histidine at position 34 instead of proline. Chicken Y2 would then not need to discriminate against the proline residue in position 34 of mammalian PP and p[Leu31,Pro34]NPY. Endogenous as well as mammalian PYY and NPY inhibited forskolin induced cAMP synthesis in a stable cell line, indicating that the divergent cytoplasmatic tail does not alter the interactions with Gi proteins. In situ hybridization detected mRNA expression in the hippocampus, which is a site of Y2 in mammals, indicating that the neuroanatomical distribution is conserved between birds and mammals.

PAPER II A neuropeptide Y receptor Y1-like gene from an agnathan, the European river lamprey: a potential ancestral gene This article reports the isolation of an NPY receptor from the cyclostome Lampetra fluviatilis. This is the first NPY receptor to be cloned from an agnathan, contributing information from a key lineage in evolution and an interesting model organism in neuroscience, and further clarifies the expansion of the NPY receptor family. The receptor clone was generated by PCR cloning using degenerate primers based upon the amino acid

27 sequences of all Y1 subfamily receptor. The full-length clone was subsequently isolated by hybridization of a gridded genomic cosmid library with a 32P-labeled probe. The lamprey Y receptor sequence exhibits a higher degree of identity to all of the Y1 subfamily receptors than to Y2 or Y5. Several methods of tree construction place the lamprey sequence within the Y1 subfamily. The analysis suggests further that the lamprey Y receptor belong in the clade containing Y4, Ya, Yb and Yc. Due to the large evolutionary distance between the lamprey and the gnathostomes and the assumed short time interval between the divergence of these animal groups and the gene duplication events it is difficult to define exactly which of these subtypes the lamprey Y receptor represents. Despite these problems the phylogenetic analyses most likely indicate that the lamprey Y receptor is a pro-orthologue to Y4 and Yb/c. The inclusion of the lamprey sequence in the phylogenetic analyses appears to have assisted in defining the evolutionary positions of other sequences. Specifically, Yb/c appear to correspond to the fourth Y1-like copy not yet found in tetrapods. The Ya receptor may have a common origin with the tetrapod Y4 subtype, or possibly even be the teleost orthologue of Y4. Expression of the lamprey receptor in a cell line facilitated binding of mammalian as well as endogenous lamprey NPY family peptides. The broad pharmacological profile of the lamprey receptor is reminiscent of the zebrafish Ya receptor, but distinct from Yb and Yc, which supports a closer relationship with the Y4/Ya lineage. Analysis of mRNA expression performed by reverse transcriptase PCR indicated the presence of transcripts in the CNS as well as several peripheral tissues.

PAPER III The neuropeptide Y receptor Y1 subfamily in the spiny dogfish, Squalus acanthias Apart from the Y receptors found in mammals, several apparent Y1 subfamily receptors have been previously reported from amphibian, avian and teleost lineages, in addition to the single Y1-subfamily receptor from an agnathan (paper II). In order to clarify the relationships of these sequences to the mammalian genes we undertook the isolation of Y1 subfamily receptors from another basal vertebrate lineage. This paper reports the first isolation in any non-mammal of orthologues corresponding to all three mammalian subtypes, namely the shark Squalus acanthias. Degenerate PCR primers based upon the amino acid sequences

28 of all Y1 subfamily subtypes were used in PCR reactions with Squalus genomic DNA. Due to the fact that no Squalus genomic or cDNA libraries were available, full-length sequences were generated using inverse PCR technique. Phylogenetic analysis with other NPY receptor sequences confirmed their positions as orthologues, as well as more clearly positioning the teleost Y1 subfamily receptors in evolutionary trees. In addition to providing the first apparently complete Y1 subfamily in a non-mammalian species, the cloning of the three individual shark genes imply important breakthroughs in the elucidation of the evolution of NPY receptors. First of all these data confirm that all three of these Y1-subfamily genes arose before the divergence of cartilaginous fishes from the teleosts and tetrapods. Thus, these duplications occurred very early in vertebrate evolution, in agreement with HOX cluster data (Holland et al., 1994). SqY4 is of particular interest since it is the first Y4 receptor to be cloned from a species in which PP is not found. Since Y4 in mammals primarily binds PP, and PP is found exclusively in tetrapods, the functional role of Y4 in non-tetrapod species will likely differ considerably from that of mammals. SqY6 is the first y6 orthologue to be cloned from a non-mammalian species, providing insights into the original functions of this subtype. Y1 was the first NPY receptor to be isolated and has been cloned in mammals, chicken and frog. Despite considerable efforts Y1 has not been isolated from any teleost, although a Y1 candidate gene has been cloned in the holostean fishes bichir and sturgeon (Larson, E.T., personal communication). The SqY1 is the first piscine Y1 orthologue to be reported. The genomic sequence contains an intron at the same position as all other Y1 genes, although expanded compared to that of tetrapods. This indicates that Y1 may have been lost in teleosts. RT-PCR detected mRNA transcripts of the shark genes in several tissues. The expression pattern appears to differ considerably from that seen in mammals. Considering the differences in putative receptor function discussed above this should not be considered surprising. Y1 in mammals is expressed in the CNS but was not detected in shark brain RNA preparations but instead in liver and kidney samples. SqY4 expression differed from that for mammalian Y4 being found primarily in retina and liver as well as brain and muscle. Transcripts of the Y6 gene, which appears to be functional judging from the deduced amino acid sequence, were detected in the retina, gastrointestinal tract, and kidney.

29 PAPER IV Cloning and Characterization of a Novel Neuropeptide Y Receptor Subtype in the Zebrafish The zebrafish has become the model organism of choice for the teleost lineage during the past decade. Together with the fact that NPY and PYY had been isolated from this species by members of our laboratory made the zebrafish a desirable organism in which to clone further NPY receptors. In order to do so degenerate primers were designed with respect to all known Y1 receptor sequences. A PCR-product found to have sequence similarity with NPY receptors was found and used as a probe to screen a zebrafish genomic and a cDNA library. The full-length sequence revealed a putative receptor, of 375 amino acids not containing any intron sequence. The receptor sequence exhibited a 50% identity to mammalian Y1 and 52% to Y4. This was much lower than expected for a teleost orthologue to either of these subtypes, suggesting that the receptor represents a novel receptor subtype, hence designated Yb. The receptor was expressed in a cell line and the pharmacological profile was investigated using iodinated porcine PYY, which bound to the receptor with picomolar affinity. Both zebrafish and porcine NPY and PYY inhibited binding of the radioligand. The affinity for zNPY was approximately ten-fold higher than for zPYY. With respect to binding of truncated NPY peptides, Yb resembles the pharmacological profile of mammalian Y1, although it did not bind either of the Y1 specific non- peptidergic ligands BIBP3226 and SR120819A. RT-PCR revealed messenger RNA transcripts of the Yb gene in brain, intestine and eye. The sequence divergence of the Yb gene along with the pharmacological profile indicated at the time that Yb represented a novel subtype. This was subsequently confirmed by the inclusion of the lamprey and shark sequences reported in paper II and III, respectively, as well as the two other zebrafish receptor sequences Ya and Yc.

30 DISCUSSION

Chicken Y2 Y2 receptors in mammals exhibit a slow rate of evolution. The Y2 receptor in chicken appears to be evolving at a similar rate exhibiting a 75-80% amino acid identity with mammalian Y2 sequences, leaving no doubt that the receptor reported in paper I indeed is the Y2 orthologue in chicken. The cytoplasmatic tail of the chicken receptor did show a higher divergence than the rest of the receptor, initially raising the question as to whether chY2 intracellular signaling differed from mammalian Y2. An assay for inhibition of cAMP synthesis revealed that this was not the case and that interactions with Gi-proteins were similar to Y2 receptors in other species. The chY2 receptor displayed similarities with its mammalian orthologues with respect to pharmacological profile and expression pattern in the brain, although some important differences were observed. One difference was that chY2 did not bind the non-peptidergic ligand BIIE0246, which has been shown to bind Y2 in mammals. Given the evolutionary distance between mammalian and avian lineages and the fact that BIIE0246 was designed to bind to human and rat Y2 this difference may be considered reasonable. More remarkable was that the receptor was found to bind p[Leu31,Pro34]NPY. This modified NPY peptide has two alterations that render it more similar to PP. Most avian PP sequences on the other hand do not have a proline residue at position 34, and it is possible that there is no evolutionary reason for chY2 to discriminate against the proline in p[Leu31,Pro34]NPY. The information gained from the binding experiments in paper I have proved useful in mutagenesis studies of the human Y2 receptor. As chY2 did not bind the Y2 specific antagonist BIIE0246, positions in the chicken receptor sequence corresponding to residues in human Y2 with postulated ligand binding functions were mutated to become identical to the human sequence. Several of the mutated chicken receptor variants revealed increased affinity for the BIIE0246 enabling the identification of these residues as vital for the ligand-receptor interaction (Berglund, M.M et al, personal communication). This clearly illustrates an advantage of studying receptors from an evolutionary perspective.

The teleost receptors The three Y1-subfamily receptors isolated from zebrafish and one from cod were at first confusing, not appearing to be orthologues to any of the subtypes isolated from mammals. With the subsequent isolation of receptors

31 from other basal vertebrate lineages the position of the teleost receptors have become clearer, although some questions still remain. One reason for the complicated NPY receptor situation in teleosts may be the additional genome duplication proposed for the teleost lineage resulting in additional gene duplicates. Zebrafish Yb along with Yc and cod Yb now appear as likely descendants of the fourth postulated Y1-like gene arising from the early vertebrate duplication event. This gene seems to have been lost in other tetrapods. The isolation of these teleost subtypes along with the shark and lamprey sequences in papers II and III have added considerable evidence for the theory of two large-scale duplications having occurred early in vertebrate evolution. Even the position of Ya as a possible Y4 duplicate seems more probable as a result of the isolation of multiple NPY receptors in vertebrates more distantly related to mammals.

The Y1 subfamily in the spiny dogfish The cloning of three Y1-like sequences in the shark, Squalus acanthias, and their identification as orthologues to the three known mammalian Y1 subfamily receptors revealed that the genomic organization of elasmobranchs resembles that of mammals with regard to the Y receptors. This is of great evolutionary importance considering that the repertoire of Y1-like receptors in teleosts differs considerably. Not only are the shark receptors from a species which is more distant to mammals than the teleosts, but this was the first reported isolation in any non-mammal of orthologues corresponding to all three mammalian subtypes. The sequence and phylogenetic information acquired from this study provides additional evidence for two large-scale or total genome duplications early in vertebrate evolution, prior to the split of elasmobranchs form the vertebrate tree. The gene coding for SqY1 contains an intron at the same position as all other Y1 genes indicating that this intron was introduced to the gene shortly after the second duplication event, and should be expected to be found in all other gnathostomes. SqY4 is the first true orthologue of the mammalian Y4/PP1 receptor to be cloned from a species without endogenous PP. It appears that after the inclusion of the SqY4 sequence in phylogenetic trees zebrafish Ya could possibly be a Y4 duplicate, but this remains to be confirmed. The role of Y4 in the non-tetrapod gnathostomes is therefore still unknown. Considering this, it is interesting that SqY4 exhibits such a high degree of sequence identity to mammalian Y4. As y6 seems to be redundant in mammals, SqY6 should prove valuable in defining the role of Y6 in the other vertebrate classes, and it will be of great

32 interest to understand why it has become redundant in mammals. Preliminary information from chicken and the marsupial wallaby (Salaneck et al., unpublished) indicates a likely functional status for Y6 in these lineages, strengthening the case for Y6 having an important physiological role in many vertebrate species. The expression pattern of the Y1 subfamily genes in Squalus detected by RT-PCR indicate functional differences of the subtypes as compared to the mammalian orthologues. If the three subtypes arose due to the second genome duplication it would seem likely that the subtypes had not yet diverged or acquired their functional characteristics at the time of the chondrichthyan/gnathostome split, allowing this to happen separately in the two evolutionary branches. It would be of great evolutionary interest to investigate how orthologous genes acquire different functions while at the same time retaining a high degree of sequence identity.

The lamprey Y receptor The lamprey receptor described in paper II was isolated from the class of chordates most distantly related to the mammals. The lampreys are a crucial class to study as they diverged from other vertebrates prior to a proposed second large chromosome or genome duplication event in the gnathostome lineage. Although it was difficult to completely resolve the position of the lamprey NPY receptor, several methods of phylogenetic analyses generated trees with identical or nearly identical topology placing the lamprey Y receptor sequence basal to Y4 and all teleost receptors. This indicates that the LfY receptor could indeed be a descendent of the ancestral receptor to the gnathostome Y4 and Yb/c subtypes, i.e. the pro-orthologue of these. This could be confirmed if a Y1/Y6 pro-orthologue was isolated in this species. Considering the resolution in the phylogenetic analyses, and the great evolutionary distance of the lamprey from tetrapods it was of utmost importance to investigate the pharmacological properties of the lamprey NPY receptor. This was facilitated by the previous isolation of endogenous NPY family peptides from the lamprey. Of further interest, an NPY family peptide unique to the lamprey lineage, PMY, was also available for these experiments. Despite the great evolutionary distance, iodinated pPYY was found to bind LfY with picomolar affinity. Binding of the radioligand was effectively inhibited by all three endogenous peptides LfPYY, LfNPY and LfPMY as well as by pNPY, pPYY and truncated pNPY peptides. The highest affinities were exhibited by the three lamprey peptides.

33 RT-PCR detected mRNA transcripts in the CNS as well as in peripheral tissues, further indicating an important role for the receptor in the NPY system in lampreys. As previous neurological studies have been performed in the lamprey it could be of interest to further investigate the physiological functions of LfY as well as attempt the isolation of other NPY receptors in this species.

NPY receptors in vertebrates Although much remains to be done before we can completely understand the events that shaped the human genome and the genomes of our vertebrate relatives, it is clear that gene duplications is one of the most important evolutionary mechanisms involved in these processes. The results presented here help clarify the early evolution of the NPY receptor family. The results support the notion of two large-scale duplications having occurred very early in vertebrate evolution. Possibly these extensive gene duplications provided the prerequisite for the evolution of the complex anatomy of gnathostomes and their rapid divergence. The Y1 subfamily arose by means of chromosomal duplications and the data presented here show that these took place before the divergence of gnathostomes, more than 420 MYA (paper III). The schematic diagram in figure show a possible way in which the Y1 subfamily may have evolved, resulting in the different repertoires of Y1-like receptors in major vertebrate lineages. The scheme is speculative as we may quite possibly find additional receptors in some of these lineages. The situation is still unclear for teleosts why more data is needed from representatives from this lineage. The three Y1 subfamily receptors found in the shark are orthologues to the mammalian subtypes. This could means that the ancestral gene of the fish Yb/c has been eliminated at least twice, both in the tetrapod lineage and the elasmobranch lineage. However, it remains possible that this gene is present in these lineages but has not yet been discovered. As it appears that gene death occurs frequently after duplications or tetraploidizations, it would not be unlikely that the Yb/c gene may have been lost at two separate occasions in these lineages.

34 Mammalia Aves Chondrostei Teleostei Chondrichthyes Agnatha

human chickenbichir sturgeon zebrafish shark lamprey Y1y6 Y4 Y1Y6 Y4 Y1 Y1 Ya YbYc Y1Y6Y4 Y4/b

? t g in s is m r o d n u s fo e t at e d y r t o III o h N c to ro p n i t d te g c in e ss xp i E m r o d n u fo t e y t o N

II I

Y1 family ancestor

Fig 7. A hypothetical scheme for the evolution of the Y1-subfamily in major chordate lineages, based on the 2R theory and the results presented in this thesis.

35 FUTURE PERSPECTIVES

The theory of two large-scale or genome duplications early in vertebrate evolution provides a plausible explanation for the existence of many vertebrate gene families. Opponents to this theory have largely based their arguments on the phylogenetic analysis of a few gene families, and subsequent failure to find two pairs of genes within families of four. Other skeptics have merely searched for gene families comprised of four members, concluding that many gene families consisting of three or five genes argue against the 2R hypothesis (Hughes, 1999; Li et al., 2001). As I hope to have shown in this thesis, phylogenetic analysis alone is not sufficient to resolve relationships of species that diverged in early vertebrate evolution, especially if there are large differences in rates of evolution between the genes. A deeper investigation of chromosomal organization of the genes as well as the functions of gene products is essential to understand the events that shaped the genomes of diverse vertebrate lineages. Loss of gene function due to mutations after duplication events is well documented (Nadeau & Sankoff, 1997; Postlethwait et al., 2000). Also, more recent gene duplications, either due to large-scale or local events, make it difficult to evaluate the 2R theory merely by counting gene family members. By obtaining more detailed information concerning chromosomal localization, anatomical distribution, and functional properties of genes, much firmer conclusions can be drawn concerning their evolution. A further advantage to performing more detailed analysis of the genes and their products is that information can be obtained that can be utilized in understanding the physiology or pathology associated with the investigated genes. In this work, we have provided additional information concerning the early evolution of vertebrates as well as elucidating receptor-ligand co- evolution. The physiological effects mediated by the NPY peptide-receptor system appear to be involved in the complex systems associated with several pathological conditions, including weight disorders and high blood pressure. A major step in the design of novel drugs to treat these conditions is understanding ligand-binding mechanisms. One efficient way of achieving this is by comparing the pharmacological properties of homologous receptor subtypes in different species. This approach will surely be employed to a greater extent as we acquire more sequence and physiological information from our vertebrate relatives. In the near future, the cloning of additional NPY receptors could assist in both of the aspects above. In the agnathan lamprey, we have cloned

36 a receptor gene that appears to be a pro-orthologue to the gnathostome subtypes Y4 and Yb/c. We therefore expect to find a Y1/Y6 pro-orthologue in the lamprey. The sequence information gathered here should assist in the continued search for this and other candidate NPY receptor genes. Considering that relatively few nuclear genes have been sequenced in the agnathan fishes, a complete investigation of the NPY receptor family in lamprey and hagfish could greatly improve our understanding of agnathan inter-relationships. Information from other gene families, such as the HOX gene clusters, suggests that protochordates should have a single chromosomal segment harboring NPY receptor genes. Cloning of NPY receptor genes in a protochordate or even an echinoderm would help resolve the evolutionary past of the gene family. If we should eventually find these pre-vertebrate genes, it will then be of great interest to investigate and date the events that generated the three presumed ancestral receptor genes, corresponding to Y1, Y2 and Y5, from a presumed original NPY receptor pro-orthologue. The chicken Y2 receptor described in paper I provides an illustrative example of how comparative biology can be applied to the understanding of receptor–ligand interactions. By performing site-directed mutagenesis experiments with chicken Y2 we have gained important insights to the interactions of human Y2 and the Y2-selective antagonist BIIE0246 (Berglund, M.M., personal communication). Understanding binding, especially that of non-peptidergic ligands such as BIIE0246, is an essential step in the design of improved pharmacological therapy, as peptides have severe therapeutic limitations due to short half-lives. Our field of research, and our comparative approach, may assist in the development of drugs for the treatment of anorexia, bulimia and hypertension. This clearly illustrates the virtue of comparative reasoning in the field of medical science. A decade ago, computer based drug design was predicted to increase the efficiency of which novel substances could be developed and applied to clinical use. Unfortunately, the expected progress in this field has proved disappointing so far. However likely that computer assisted drug development will become more and more successful in the future, the method described above appears to be an inexpensive and elegant alternative and definitely a valuable addition to this area of science. Hopefully this can be applied to research concerning many other pathological conditions, and not only those more specific to the industrialized and wealthy world.

37 CONCLUDING REMARKS

Three major contributions to the field of science that have had a profound influence on our view of the origins of life were mentioned in the introduction. As I complete this thesis, a fourth is nearing its completion. The sequence of the entire human genome will shortly be accessible for the public. Contrary to the contributions made by a few individuals such as Darwin, Wallace, Mendel, Watson and Crick, the HUGO project has been carried out by an unprecedented cooperation of large parts of the scientific community. The information unveiled by this project will undoubtedly accelerate the speed at which we will be able to isolate additional receptors as well as more generally increase our understanding of life and evolution.

38 ACKNOWLEDGMENTS

The following people have contributed to the completion of this thesis and will be acknowledged in Swedish:

Professor Dan Larhammar, för handledning och för att ha gett mig möjligheten att genomföra detta projekt samtidigt som jag ängnat mig åt min skråutbildning. Professor Lars Oreland, för utmärkt ledning av avdelningen för medicinsk farmakologi och för goda (?) råd gällande det medicinska hantverket och racketsport. Alla nuvarande och före detta medarbetare i NPY-gruppen och avdelningen för medicinsk farmakologi för en trevlig arbetsmiljö. Mina medförfattare (My co-authors): Earl (Örl) Larson, Robert Fredriksson, Sara Holmberg, Magnus Berglund, Tim Boswell, Mike Conlon, Paula Starbäck, Don Gehlert, and Ingrid Lundell. Ytterligare tack till Örl för kritisk granskning av alla manuscript och avhandlingen, och samarbetet med rump-sparkande PCR-primrar, och Robert, Sara och Magnus för nära samarbete på lab. Christina Bergqvists insatser i de senaste terminernas telefon- och fjärrforskning har varit ovärderliga. (Jag ringer efter röntgenronden.)

BMC-biologerna: Mats F, Mats N, Peter, och Magnus. För att ni var på BMC, och vet exakt vad det innebär. Tack pojkar. GE JÄRNET MAGNE! Biologerna som höll sig ifrån BMC, för att ni höll er ifrån BMC. Grattis. Henrik! För att ha kört monstertruck genom hela utbildningen. Erik, Malin, Kalle och alla “västgötar”: goda vänner växer i små lustiga tuvor. Fredrik: jäst och 37° blir perfa. Alla i leprakolonin. Västgöta Nation i Upsala, den stora sub-familjen, och speciellt: VÄSTGÖTA NATIONS MANSKÖR som andats så mycket klokhet genom seklerna. Vive le Meauxs, och alla Aves och Pisces (Clupea sp. et c.) och andra djur! Venez enfants!

Lillasyster och Claes. Den bästa lillasystern och Claes jag någonsin haft. Anna! Tack för att du stod ut med det här! Jag hade inte gjort det utan dig! Till sist, M+P, mina föräldrar. Detta hade verkligen inte gått utan er! Tack!

Absent friends. Ruhe sanft.

“Så här roligt har jag inte haft sen de draggade efter svärmor i fel sjö.” N.D.

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